The role of human umbilical cord mesenchymal stem cells-derived exosomal microRNA-431-5p in survival and prognosis of colorectal cancer patients

Abstract We aim to discuss the role of miR-431-5p in colorectal cancer (CRC) progression via regulating peroxiredoxin 1 (PRDX1). miR-431-5p and PRDX1 expression were detected in CRC tissues and cells, and the relationship between miR-431-5p expression and prognosis of CRC patients was analyzed. Exosomes were extracted from human umbilical cord mesenchymal stem cells (hUCMSCs) and co-cultured with LoVo cells. MTT assay, flow cytometry and Transwell assay were implemented to test cell viability, apoptosis and invasion and migration ability, respectively. The tumor growth was determined as well, and the binding relation between miR-431-5p and PRDX1 was confirmed. miR-431-5p was downregulated and PRDX1 was upregulated in CRC, and miR-431-5p downregulation was associated with poor prognosis. hUCMSC-Exos suppressed the malignant behaviors of LoVo cells, and overexpression of miR-431-5p further aggravated the inhibitory effect of hUCMSC-Exos on LoVo cells. hUCMSC-Exos inhibited PRDX1 expression via miR-431-5p. PRDX1 was targeted by miR-431-5p. miR-431-5p serves as a prognostic biomarker in CRC, and hUCMSC-Exos transfer of miR-431-5p decelerates CRC cell growth by inhibiting PRDX1.


Introduction
Colorectal cancer (CRC) is a common cause of cancerassociated death [1]. The underlying mechanisms of CRC are multifactorial. Risk factors include age, gender and lifestyle, and potential genetic factors that have little influence [2]. Despite improvements in screening, addressability, and awareness, a quarter of cases are still diagnosed with advanced CRC [3]. Multi-model treatment strategies could improve the overall survival (OS), but severe side effects such as weight loss accompany [4]. Thus, novel targets are urgently needed for CRC treatment.
Exosomes are extracellular vesicles [5] that are involved in multiple physiological and pathological processes [6]. Human umbilical cord mesenchymal stem cells (hUCMSCs) have great advantages of readily available source, self-renewal property, and immunomodulation [7]. It has been identified that exosomes contain microRNAs (miRNAs), mRNAs, DNA, and proteins [8]. miRNAs can suppress gene expression by mRNA degradation and translational suppression [9]. It is known that miR-4319 [10], miR-107 [11], and miR-431 could affect CRC progression [12], while the role of miR-431-5p remains largely unknown. Peroxiredoxins (PRDXs) are essential antioxidant proteins constituting the potent defense system to sustain redox balance by converting hydrogen peroxide to water. PRDX1 is a 23-kDa stress-triggered macrophage redox protein [13] that has been addressed to be related to tumor metastasis, angiogenesis [14], and inflammation in CRC [15]. We aim to identify the role of miR-431-5p conveyed by hUCMSC-Exos in the progression of CRC via regulating PRDX1, and inferred that hUCMSC-Exos may upregulate miR-431-5p to restrict the CRC cell growth via inhibiting PRDX1.

Ethics statement
Written informed consent was acquired from all patients before this study. The protocol of this study was confirmed by the Ethics Committee of the Dongzhimen Hospital, Beijing University of Traditional Chinese Medicine. Animal ex-

Study subjects
CRC patients (n = 101) accepted resection in the Dongzhimen Hospital, Beijing University of Traditional Chinese Medicine were selected. All the patients were pathologically confirmed as primary adenocarcinoma and have not been treated with radio-or chemotherapy. Tumor and adjacent normal tissues were resected, frozen, and preserved at −80°C. OS was calculated from the resection to death time or the last follow-up visit.

Cell culture, transfection and treatment
Human CRC cell lines (Caco-2, SW480, SW620, LoVo, and HCT 116) and normal epithelial cells NCM460 were obtained from BeNa Culture Collection (Beijing, China) and cultured in specific medium. hUCMSCs was acquired from BeNa Culture Collection and cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (P/S).

Isolation and identification of hUCMSC-Exos
To eliminate interference from FBS-derived exosomes in the cell culture medium, the FBS was centrifuged at 100,000 × g for 18 h. The culture medium was replaced with medium containing 10% exosome-free FBS. After 48 h of incubation, the cell supernatant was collected. The collected supernatant was centrifuged at 500 g for 15 min at 4°C to remove cell debris, and followed by centrifugation at 2000 to remove apoptotic vesicles and centrifugation at 10 000 g for 20 min at 4°C to remove large vesicles. The supernatant was then filtered by a 0.22-μm filter, centrifuged at 110 000 g for 70 min at 4°C, resuspended with PBS, and then centrifuged in the same condition. The downstream experiments were performed by further superseding with 100 μl sterile PBS. The morphology of exosomes was observed by a transmission electron microscope (TEM). The exosomes were diluted and dropped onto the copper mesh for staining with 3% sodium phosphotungstate solution (pH = 6.8). The particle size of exosomes was measured by Zetasizer Nano ZS90 (Malvern Instrument, Malvern, USA). Western blot analysis was used to detect CD9 (1:1000), CD81 (1:1000), and TSG101 (1:1000; Abcam).

Transwell assay
Migration assay: 0.2 ml cells (concentration: 2 × 10 5 /ml) were seeded into Transwell (Corning, NY, USA) apical chambers and incubated with 100 μl serum-free medium. The basolateral chambers were added with medium containing 20% FBS and incubated for 16 h. The migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.
Invasion assay: apical chambers were coated with Matrigel (BD Biosciences) and other steps were the same as migration assay.

Flow cytometry
Apoptosis of LoVo cells was determined by flow cytometry as previously described [16] and the results were analyzed by a flow cytometer (BD Biosciences).

Western blot analysis
Proteins were performed with gel electrophoresis and transferred onto membranes, which were blocked with 5% skim milk powder and incubated with primary antibodies PRDX1 (1:1000) and GAPDH (1:1000; Abcam) and with relative secondary antibody. After development by enhanced chemiluminescent reagent, bands were exposed on the Image Quant LAS 4000C gel imager (General Electric Company, USA).

Subcutaneous tumorigenesis in nude mice
Twenty-four nude mice (aged 4-6 weeks) were randomly assigned into 4 groups (6 mice per group). LoVo cells (1 × 10 6 cells/mouse) were injected subcutaneously into the flank of nude mice. Meanwhile, a dosage of 5 mg exosomes administered into mice via tail vein injection once every 3 days for 2 weeks. Tumor size was measured with calipers after the tumor cell injection once every 7 days for a total period of 4 weeks. The tumor volume = length × width 2 × 0.5. Mice were euthanized on the 28th day of the tumor cell injection and the xenografts were isolated and weighed.

Statistical analysis
All data analyses were conducted using SPSS 21.0 software (IBM, NY, USA). The measurement data were expressed as mean ± standard deviation. The t-test was performed for comparisons between two groups, analysis of variance (ANOVA) was used for comparisons among multiple groups and Tukey's post hoc test was used for pairwise comparisons after ANOVA. Chi square test was carried out to analyze the relationship of miR-431-5p expression clinicopathological characteristics of CRC patients. Kaplan-Meier method was used to analyze the survival and Pearson test was employed to assess the correlation between miR-431-5p and PRDX1. P-value < 0.05 was indicative of statistically significant difference.

Results
miR-431-5p is downregulated in CRC and is related to the poor prognosis of CRC patients miR-431-5p downregulation has been previously identified in cancers [12,17]. To assess the role of miR-431-5p in CRC, its expression in tissues was determined. We found that CRC tissues had lower miR-431-5p expression than adjacent normal tissues (Fig. 1A). miR-431-5p expression in cells was also evaluated and it was discovered that miR-431-5p was downregulated in five CRC cell lines. The most significant downregulation of miR-431-5p expression was observed in LoVo cells, which were thereby selected for follow-up experiments (Fig. 1B).
CRC patients were separated into high and low miR-431-5p expression groups based on the median miR-431-5p expression to assess the relation between miR-431-5p expression and clinicopathological characteristics of CRC patients (n = 51). The results indicated that low miR-431-5p expression was related to tumor-node-metastasis (TNM) stage, lymph node metastasis (LNM), and differentiation degree (Supplementary Table 2). Moreover, we found from Kaplan-Meier method that patients with low miR-431-5p expression had a shorter OS (Fig. 1C).

hUCMSC-Exos suppress CRC cell growth
The surface markers of hUCMSCs were determined and we found that CD90 and CD105 were positive while CD14, CD19, and CD34 were negative ( Fig. 2A). It was observed under a TEM that hUCMSC-Exos were spherical or ellipsoidal vesicles with complete envelope and similar morph-  (Fig. 2B). It was observed by the Zetasizer Nano ZS90 that the particle diameter ranged from 30 to 120 nm (Fig. 2C). Moreover, the protein expression of CD9, CD81, and TSG101 was higher in hUCMSC-Exos than in hUCMSCs (Fig. 2D).
PKH26-labeled exosomes were co-cultured with LoVo cells and it was observed under a fluorescent microscope that green fluorescence appeared in the cytoplasm, suggesting the successful uptake of exosomes by LoVo cells (Fig. 2E). To further explore the transfer of miR-431-5p, we electrotransferred FITC-miR-431-5p (green) into exosomes from hUCMSCs, added Dil tags (red), and incubated LoVo cells for 48 h. Co-localization of FITC and Dil was observed in the receptor LoVo cells, indicating that the FITC-miR-431-5p-containing cellular exosomes were internalized by LoVo cells (Fig. 2F). miR-431-5p expression in LoVo cells was assessed and we found that miR-431-5p was upregulated with hUCMSC-Exos treatment (Fig. 2G). It was found in MTT assay, Transwell assay, and flow cytometry that hUCMSC-Exos suppressed proliferation, migration, and invasion of LoVo cells; results of flow cytometry implied that the exosomes promoted LoVo cell apoptosis (Fig. 2H-J).
PRDX1 downregulation reverses the effects of miR-431-3p downregulation on LoVo cells PRDX1 expression in LoVo cells was assessed and we observed that compared with the miR-431-5p inhibitor group, PRDX1 expression was decreased in the miR-431-5p inhibitor + si-PRDX1 group (Fig. 5A). Biological functions of LoVo cells were determined and it came out that si-PRDX1 abolished miR-431-3p inhibitor-induced effects on LoVo cells (Fig. 5B-D).

Discussion
CRC has a higher incidence and mortality in developed countries [19]. We found that hUCMSC-Exos upregulated miR-431-5p to restrict CRC cell growth via inhibiting PRDX1.
LoVo cells were treated with HUCMSC-Exos to clarify their effects on GC cell growth, and we found that hUCMSC-Exos were able to suppress the growth of CRC cells. Similarly, it has been discovered that hUCMSC-Exos reduced pancreatic ductal adenocarcinoma cell growth [20] and restrained malignant behaviors of breast cancer cells [21]. It has been described that extracellular vesicles derived from hUCMSCs suppressed colon adenocarcinoma cell proliferation and migration [22] and MSCs-derived exosomes inhibited angiogenesis, proliferation, and migration of breast cancer cells [23]. Moreover, this study suggested that hUCMSC-Exos could upregulate miR-431-5p expression and downregulate PRDX1 expression.
We determined miR-431-5p expression in CRC tissues and cells, and we found that miR-431-5p was downregulated in CRC. miR-431-5p has once been found to be downregulated in CRC cancer tissues and cells [24], and miR-431 was also downregulated in CRC [25]. Considering the dysregulation of miR-431-5p in CRC, we analyzed the predictive role of miR-431-5p in CRC patients, and our results implied that low miR-431-5p expression suggested a poor prognosis, and miR-431-5p expression was associated with TNM, LNM, and differentiation of CRC patients. Wu et al. have illuminated that miR-431-5p was associated with the OS of esophageal carcinoma patients [26], and miR-431 was related to LNM and TNM stage of patients with hepatocellular carcinoma [27]. Results of our experiments indicated that overexpressed miR-431-5p conveyed by hUCMSC-Exos repressed the development of CRC cells while inhibited miR-431-5p had opposite effects on CRC cells. Similarly, it has been recently illustrated that the ectopic expression of miR-431-5p repressed the proliferation and invasion of CRC cells [24], and Su et al. have validated that the migration of CRC cells transfected with miR-431 mimic was markedly reduced, while cells transfected with miR-431 inhibitor performed reverse alterations [25].
It is known that miRNAs can post-transcriptionally modulate target gene expression [28]. Our study suggested that PRDX1, targeted by miR-431-5p, was upregulated in CRC tissues and cells. Similarly, studies have uncovered that PRDX1 exhibited a high expression level in CRC [14,15]. However, the binding relationship between miR-431-5p and PRDX1 remains scarcely discussed. Furthermore, we discovered that PRDX1 downregulation mitigated the impacts of downregulated miR-431-5p on CRC cells. It has been elucidated that PRDX1 inhibition could suppress the growth of CRC cells [18].
In conclusion, we found that miR-431-5p served as a predictive role in CRC patients' prognosis, and upregulated miR-431-5p conveyed by hUCMSC-Exos suppresses CRC progression by inhibiting PRDX1. Our study may contribute to CRC treatment, while there remains much to do to reveal the underlying mechanisms.

Supplementary data
Supplementary data is available at Mutagenesis online.

Conflict of Interest Statement
None declared.